Different micro-organisms differentially induce Arabidopsis disease response pathways

Plant Physiol. Biochem. 39 (2001) 673−680 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942801012827/FLA

Different micro-organisms differentially induce Arabidopsis disease response pathways Bart P.H.J. Thommaa*, Koenraad F.M. Tierensa, Iris A.M.A. Penninckxa, Brigitte Mauch-Manib, Willem F. Broekaerta§, Bruno P.A. Cammuea‡ a

Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B3001 Heverlee, Belgium

b

Department of Biology/Plant Biology, University of Fribourg, 3, route Albert-Gockel, CH-1700 Fribourg, Switzerland

Received 15 January 2001; accepted 27 February 2001 Abstract – In the model plant Arabidopsis thaliana, several signal transduction pathways can be activated upon pathogen challenge leading to the activation of different (sets of) effector molecules. In the past it has been demonstrated that these different signal transduction pathways contribute differentially to resistance against distinct microbial pathogens. In this study, it is shown that not all pathogens activate the full set of defence responses. This indicates that depending on the particular interactions between elicitors and suppressors with their cognate plant targets, defence response cascades may or may not become activated during pathogenesis. These findings imply that current models of plant-pathogen interactions must be revised to take into account the pathogen-dependent nature of many defence responses. © 2001 Éditions scientifiques et médicales Elsevier SAS Arabidopsis thaliana / camalexin / plant-pathogen interaction / PR-protein PR, pathogenesis-related / SA, salicylic acid

1. INTRODUCTION Plants have developed inducible defence systems that can be activated upon detection of a potential pathogen. During the last decade, Arabidopsis has become a very important model plant to unravel how these defence systems can control pathogen attack. This is largely due to the availability of many mutants in different defence response pathways. Studies using these mutants have led to a reasonable insight in how different defence pathways are attributed to control different pathogens (reviewed in [24]). A defence component that is produced only at the site of attempted pathogen ingress is the phytoalexin *Correspondence and reprints: fax +32 16 321966. E-mail address: [email protected] (B.P.H.J. Thomma). § Present address: CropDesign N.V., Technologiepark 3, B-9052 Gent, Belgium. ‡ Member of the Flanders Interuniversity Institute for Biotechnology-VIB

camalexin [25]. Using the phytoalexin-deficient Arabidopsis mutant pad3-1 which is completely blocked in its ability to synthesise camalexin due to a frameshift mutation in the biosynthetic gene PAD3 [33], camalexin has been demonstrated to be required for full resistance against the fungus Alternaria brassicicola but not against the fungus Botrytis cinerea or the bacterium Pseudomonas syringae [10, 23]. Apart from locally induced defence responses, plants can also induce defence responses in tissues distant from the initial infection site. Salicylic acid (SA) has been recognised as a central molecule in these so called systemic defence responses. In Arabidopsis, the SA-dependent defence pathway results in the induction of at least the pathogenesis-related (PR) genes PR-1, PR-2 and PR-5 [26, 27] and SA-dependent signalling has been shown to be required for resistance of Arabidopsis plants to at least the pathogens Ps. syringae, Peronospora parasitica and Erysiphe orontii [2, 3, 5, 18]. It has become clear, however, that SA is not the only signalling molecule involved in mounting disease resistance responses. Additional to a

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SA-dependent defence response pathway, a jasmonate/ethylene-dependent, yet SA-independent, defence response pathway exists in Arabidopsis resulting in the induction of a different subset of PR-proteins comprising at least PR-3, PR-4 and PR-12 (PDF1.2) [16, 21]. Arabidopsis mutants that are insensitive to either jasmonate (coi1-1) or ethylene (ein2-1) have been shown to display enhanced resistance towards the fungus B. cinerea and the bacterium Erwinia carotovora but not to P. parasitica [15, 21, 22]. Additionally, it was shown that jasmonate insensitivity leads towards enhanced susceptibility towards A. brassicicola and Pythium spp. [19, 21, 30]. Remarkably, the fungus Plectosphaerella cucumerina has been shown to be controlled by jasmonate-dependent signalling as well as SA-dependent signalling [20]. The above discussed findings indicate that in Arabidopsis different defence response pathways that depend on different signalling molecules can be activated, and that these pathways differ in efficacy for controlling distinct (groups of) pathogens. This specificity can be explained in two different ways: either all pathogens trigger the production of the same defence components, but these components are not all active against a particular pathogen, or the pattern of induction of different defence components differs from pathogen to pathogen. To discriminate between both possibilities, the relative induction levels of a marker gene for the SA-dependent pathway, a marker gene for the JA-dependent pathway and the production of camalexin were assessed in comparative tests presented in this paper. The set of pathogens used to trigger the Arabidopsis defence consisted of a biotrophic bacterium (Pseudomonas syringae pv. tomato), a necrotrophic bacterium (Erwinia carotovora pv. carotovora), a biotrophic fungus (Peronospora parasitica) and three different necrotrophic fungi (Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina). Hence, these pathogens represent a broad range of possible infection modes and a wide range of taxonomic diversity.

2. RESULTS 2.1. Induction of SA-dependent and jasmonate/ethylene-dependent defence genes by different pathogens Four-week-old Arabidopsis plants were inoculated with either of the pathogens A. brassicicola, B. cinerea, E. carotovora, Pl. cucumerina and an avirulent as well as a virulent strain of both P. parasitica and Ps.

syringae pv. tomato. On wild-type plants, inoculation with A. brassicicola leads to small brown non-spreading lesions after 48 to 72 h, whereas inoculation with Pl. cucumerina causes slow spreading water-soaked lesions. Infection of B. cinerea causes necrosis of the inoculated leaves 72 to 96 h after inoculation which, in a small percentage of the inoculated plants, will spread systemically and result in total plant decay. E. carotovora causes soft rot symptoms 24 h following inoculation, leading to leaf decay approximately 4 d later. Inoculation with virulent strains of either Ps. syringae or P. parasitica results in little host damage, whereas the avirulent strains cause tissue necrosis in a hypersensitive response. Over a period of 5 d after inoculation, plant material was collected to extract RNA that was hybridised with a probe for PDF1.2 as a marker for jasmonate/ethylenedependent defence responses and with a probe for PR-1 as a marker for SA-dependent defence responses. In figure 1, it is shown that high levels of PDF1.2 transcripts can be detected within 48 h after inoculation with A. brassicicola, Pl. cucumerina, B. cinerea or E. carotovora, and to a lesser extent with P. parasitica. In the case of the avirulent Ps. syringae strain, slight expression was observed only at 72 h after inoculation, whereas no expression was observed in the interaction with the virulent strain up to 96 h after inoculation. A. brassicicola, Pl. cucumerina and B. cinerea not only caused a strong induction of the gene encoding PDF1.2, but also strongly induced PR-1 (figure 1). Remarkably, although expression of PDF1.2 is very pronounced, almost no accumulation of PR-1 transcript could be observed in the interaction with E. carotovora. Compared with PR-1 transcript levels accumulating after inoculation with A. brassicicola, Pl. cucumerina and B. cinerea, transcripts of this gene were less abundant but appeared faster in the interaction with Ps. syringae. In case of inoculation with the avirulent Ps. syringae strain, PR-1 expression could be detected even after 12 h. This early induction should probably be regarded as a consequence of the method of inoculation: whereas the fungal pathogens are applied as a spore suspension on the surface of the leaves, Ps. syringae is inoculated by forcing a bacterial suspension through the stomata into the intercellular spaces. In that way, the bacteria are directly in contact with the plant cells, whereas the fungal spores first have to germinate and subsequently penetrate the cuticle to contact the plant cell. In Arabidopsis plants inoculated with P. parasitica, expression of PR-1 occurs rather late in the interaction with a virulent

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Figure 1. Induction of pathogenesis-related genes in Arabidopsis in response to infection with different pathogens. Four-week-old soil-grown Arabidopsis plants (ecotype Col-0) were infected with A. brassicicola, Pl. cucumerina, B. cinerea strain MUCL30158, E. carotovora, Ps. syringae pv. tomato DC3000, Ps. syringae pv. tomato DC3000 carrying avrRpt2 and P. parasitica strains Noco and Emwa and harvested at 12, 24, 48, 72 and 96 h following treatment. RNA blots were hybridised with the probes indicated on top of the figure.

strain (Noco) or at a low level in the interaction with an avirulent strain (Emwa).

2.2. Production of camalexin by different pathogens Both A. brassicicola and Pl. cucumerina not only induce high levels of PDF1.2 and PR-1 induction, but they also induce high levels of camalexin accumulation (figure 2). The strongest increase was observed between 24 and 48 h, and camalexin levels further increased moderately until at least 96 h after inoculation. In case of inoculations with B. cinerea, E. carotovora or Ps. syringae, levels of camalexin accumulation were significantly lower compared to the inoculations with A. brassicicola and Pl. cucumerina, but nevertheless camalexin production was clearly induced. This in contrast to inoculation with either the virulent or the avirulent strain of P. parasitica, in which case no camalexin could be detected.

3. DISCUSSION The comparative studies presented in this paper indicate that not all pathogens activate the full set of defence responses. One striking observation is that of Erwinia carotovora pv. carotovora, which causes strong induction of PDF1.2 but no detectable levels of PR-1 transcript, thus indicating that jasmonate-dependent responses are induced while SA-dependent responses are not. In line with our observation that E. carotovora

strongly induces jasmonate-dependent defence responses, it was shown recently that plant cell wall degrading enzymes of E. carotovora induce an Arabidopsis gene with high homology to allene oxide synthase, the first enzyme after lipoxygenase in the biosynthesis pathway of jasmonates [14]. Additionally, culture filtrate of E. carotovora containing cell wall degrading enzymes has also been shown in tobacco to induce defence responses different to those induced by SA [29]. The response of Arabidopsis plants infected with E. carotovora is in contrast to the response triggered by a virulent strain of the bacterial pathogen Pseudomonas syringae. Inoculation with this pathogen leads to induction of PR-1, but not of PDF1.2. A similar observation has previously been made with the biotrophic fungal pathogen Erysiphe orontii, which induces the SA-dependent genes PR-1, PR-2 and PR-5 in Arabidopsis but not the jasmonate-inducible genes Thi2.1 and PDF1.2 [18]. On the other hand, the necrotrophic fungal pathogens Alternaria brassicicola, Plectosphaerella cucumerina or Botrytis cinerea all induce high levels of both PR-1 and PDF1.2 transcripts. At 96 h after B. cinerea inoculation, PDF1.2 and PR-1 induction is already very low. However, by that time the fungus is largely contained: the inoculated leaf is necrotised and the fungus only spreads occasionally. Also upon inoculation with a virulent as well as an avirulent strain of Peronospora parasitica, accumulation of PR-1 and PDF1.2 transcripts is observed, though the response is slower than for the necrotrophic fungal pathogens. However, since P. parasitica is a biotrophic fungus incidentally forming

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Figure 2. Relative levels of induction of camalexin accumulation in Arabidopsis wild-type plants after inoculation with different pathogens. Four-week-old wild-type plants were inoculated with the different pathogens indicated on top of the figure and camalexin accumulation was determined in plant material collected 12 (1), 24 (2), 48 (3), 72 (4) and 96 (5) h after inoculation. Data points represent averages with standard errors of measurements on three samples. The highest camalexin level was set at 100 %.

haustoria and thus not penetrating large numbers of plant cells, it is reasonable to assume that it takes more time to generate defence-related components above a certain threshold. Interestingly, in case of the compatible P. parasitica interaction, induction of PR-1 as well as PDF1.2 occurs later compared to the incompatible interaction, but a clearly higher transcript level is obtained for PR-1. This is in contrast to PR-1 induction after inoculation with an avirulent or a virulent strain of Ps. syringae pv. tomato DC3000. With this pathogen, PR-1 accumulation is triggered faster and to a higher level in the incompatible interaction than in the compatible interaction. In many cases, avirulent races or pathovars of a pathogen have been found to induce defence-related genes earlier and to a higher level in comparison to their virulent counterparts, which could be explained by an efficient recognition system allowing a fast response [4, 8, 31]. However, the opposite has been reported as well [6, 9], which can be explained by a more extensive colonisation leading to a local response which is elaborated over a more extended area. In addition, the level of camalexin accumulation varied greatly depending on the pathogen used to challenge Arabidopsis plants (figure 2). Under the

conditions used in this study, the highest levels of camalexin accumulation were obtained after inoculation with A. brassicicola and Pl. cucumerina, whereas no camalexin at all was observed in the interaction with either a virulent or an avirulent P. parasitica isolate. Likewise, Reuber et al. [18] observed that a virulent isolate of the biotroph E. orontii induces little or no camalexin. Possibly, the number of plant cells producing camalexin in the interaction with biotrophic fungal pathogens is too low to allow accumulation of camalexin up to detectable levels. In the interaction with Ps. syringae, it was observed that the level of camalexin accumulation is comparable for the avirulent and the virulent strain. This observation confirms that of Glazebrook and Ausubel [10] who have shown that not only avirulent, but also virulent Ps. syringae spp. were able to induce camalexin in Arabidopsis wild-type plants. The extreme variations in the level of induction of defence responses depending on the challenging pathogens, as observed in this and other studies [9, 13], call for care when it comes to drawing up general models of pathogen-induced defence responses. Indeed, one factor that is often not taken into account is that pathogens differ in the cocktail of elicitors and suppressors they secrete or otherwise bring in contact with

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plant cells. Depending on the particular interactions between these elicitors and suppressors with their cognate plant targets, defence response cascades may or may not become activated during pathogenesis. Previous studies based on the use of mutants affected in particular defence response pathways have pointed to the conclusion that not every defence response pathway contributes effectively to resistance against a given pathogen [11, 18, 21–23]. However, the apparent lack of contribution of a given defence response pathway to resistance against a particular pathogen can either be due to failure to mount the response upon pathogen recognition, to poor effectiveness of the defence response in halting pathogen ingress, or to both. Examples of the first possibility are the contribution of camalexin to resistance of Arabidopsis against the biotrophic fungi P. parasitica and E. orontii. The Arabidopsis mutant pad3-1 which is completely blocked in its ability to synthesise camalexin [33] is as resistant as wild-type plants to P. parasitica and E. orontii [11, 18]. One reason is undoubtedly that no or very little camalexin production is triggered upon infection of wild-type plants by either P. parasitica (this study) or E. orontii [18]. Whether or not camalexin itself is effective against any of these fungi can not be concluded from the current data. A similar case is that of the interaction between Arabidopsis or tobacco and E. carotovora. This pathogen does not activate SA-dependent genes during the interaction with wild-

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type plants ([15, 29]; this study) and SA-degrading NahG tobacco transformants are equally susceptible to this pathogen as wild-type plants [15, 28]. Hence, no conclusion can be drawn as to whether or not SA-dependent defence systems are effective against E. carotovora, as this pathogen apparently avoids triggering them. Examples of the second possibility, where a pathogen does trigger a particular defence response but a mutation in that defence pathway has no influence on the outcome of the interaction, have also been identified. B. cinerea activates production of camalexin and SA-dependent genes in Arabidopsis, yet Arabidopsis mutants which can not produce camalexin (pad3-1) or can not induce SA-dependent genes (NahG, npr1-1) are not more susceptible to B. cinerea than wild-type plants [21, 23]. Hence, the conclusion that B. cinerea might be tolerant to the action of camalexin and SA-controlled effector molecules appears to be justified in this case. Similar conclusions can be drawn for the apparent lack of effectiveness of SA-dependent genes to control A. brassicicola, of camalexin to control Ps. syringae, and of jasmonate-dependent genes to control P. parasitica. The examples discussed above as well as others are represented in a schematic way in table I. This table summarises currently available data on the resistance of Arabidopsis defence pathway mutants against different pathogens as well as on the ability of these different pathogens to activate defence pathways. This

Table I. Overview of the interactions between Arabidopsis and different pathogenic micro-organisms. The increased susceptibility of pathway mutants versus wild-type plants is based on data derived from the JA-response mutants coi1-1 and jar1-1, the SA-response mutant npr1-1 and transformant NahG and the camalexin production mutant pad3-1. A + sign indicates increased susceptibility compared to wild-type plants, a = sign indicates an equal level of resistance. The ability to trigger defence responses by different pathogens is indicated by – for no induction, +/– for very weak induction, + for moderate induction and ++ for strong induction. Effectiveness of the defence response to the pathogen is deduced from data on the increased susceptibility of defence pathway mutants together with data on the ability to trigger the defence pathways. A + sign indicates that the defence pathway is probably effective because the pathway is induced and the corresponding mutant shows enhanced susceptibility versus wild-type plants. A – sign indicates that the defence pathway is probably not effective because the pathway is induced but the corresponding mutant is not altered in susceptibility. A ? sign indicates that no conclusion can be drawn because the pathway is not induced by the pathogen. (a) indicates avirulent strain, (v) virulent strain. a N.D. is not determined. b References: 1 = [21]; 2 = [23]; 3 = [20]; 4 = [15]; 5 = [17]; 6 = [2, 3, 5]; 7 = [10, 11]; 8 = [11]; 9 = [19]; 10 = this study. Pathogen

A. brassicicola Pl. cucumerina B. cinerea E. carotovora Ps. syringae (a) Ps. syringae (v) P. parasitica (a) P. parasitica (v) E. orontii

Increased susceptibility of pathway mutant to the pathogen

Ability to trigger the response by the pathogen

Effectiveness of the response pathway to the pathogen

JA resp.

SA resp.

camalexin

JA resp.

SA resp.

camalexin

JA resp.

SA resp.

camalexin

+1b +3 +1 +4 =5 N.D. =1 = =9

=1 +3 =1 =4 +6 +6 +6 +6 +9

+2 N.D.a =2 N.D. =7 =7 =8 =8 =9

++10 ++10 ++10 ++10 +/–10 –10 +10 +10 –9

++10 ++10 ++10 –10 +10 +10 +10 ++10 ++9

++10 ++10 +10 +10 +10 +10 –10 –10 –9

+ + + + – N.D. – – –

– + – ? + + + + +

+ N.D. – N.D. – – ? ? ?

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overview clearly indicates that pathogens strongly vary in their ability to activate different defence compounds as well as in their ability to withstand different defence compounds. Full understanding of these pathogen-dependent variations will require more molecular insight in the elicitors and suppressors produced by pathogens as well as of mechanisms used by pathogens to tolerate potentially deleterious effects of plant defence compounds.

4. METHODS 4.1. Biological material Growth and spore harvesting of the fungi Alternaria brassicicola (strain MUCL20297; Mycothèque, Université Catholique de Louvain, Louvain-la-Neuve, Belgium), Botrytis cinerea (strain MUCL30158, Mycothèque, Université Catholique de Louvain, Louvainla-Neuve, Belgium), and Plectosphaerella cucumerina (provided by Dr B. Mauch-Mani, Université de Fribourg, CH) were done as described previously [1]. Peronospora parasitica strains Wela and Noco were maintained on living Arabidopsis plants of the Weiningen and Columbia ecotype, respectively. Pseudomonas syringae pv. tomato DC3000 and Ps. syringae pv. tomato DC3000 supplemented with plasmid pLH12 carrying avrRpt2 [32] were grown overnight at 28 °C in King’s B medium [12] supplemented with the appropriate antibiotics (rifampicin at 25 µg·mL–1 for Pst DC3000 and also tetracycline at 10 µg·mL–1 for the strain carrying plasmid pLH12). Erwinia carotovora (strain LMG 6663) was grown overnight at 28 °C in L-broth.

4.2. Plant inoculations Arabidopsis plants of the ecotype Col-0 (obtained from the Nothingham Arabidopsis Seed Centre) were essentially grown as described [16]. After 4 weeks, plants were inoculated with pathogens. Inoculation of Arabidopsis plants with A. brassicicola or Pl. cucumerina was performed on 4-week-old soil-grown plants by placing two 5-µL drops of a suspension of 5·105 conidial spores·mL–1 in water on each leaf. For the inoculation with B. cinerea, two needle-prick wounds were applied to the leaves and the fresh wounds were covered with 5 µL drops of a suspension of 5·105 conidial spores·mL–1 in 1.2 g·L–1 potato dextrose broth (Difco, Detroit, USA). Inoculation with P. parasitica was done by spraying until droplet run-off with a suspension of 105 conidial spores·mL–1 in

water. All control plants were mock-inoculated with water, except for B. cinerea, for which the wounds of control plants were covered with 5 µL drops of 1.2 g·L–1 potato dextrose broth. For inoculations with Ps. syringae and E. carotovora, bacteria were grown overnight in the appropriate medium. The OD595 was determined, cells were pelleted and suspended in 10 mM MgCl2 to reach a final OD595 of 0.04 or 1.0 for Ps. syringae and E. carotovora, respectively. Ps. syringae was inoculated by pressing a 1-mL syringe without a needle against the abaxial side of the leaves and forcing the bacterial suspension through the stomata into the intercellular spaces [10]. For inoculation with E. carotovora, two 5-µL drops of the bacterial suspension were placed on each leaf. All inoculated plants were incubated at 100 % relative humidity in propagator flats covered with clear polystyrene lids in the growth chamber for the time periods indicated.

4.3. Induction of defence reactions The method for camalexin detection was performed as described [23]. For testing the induction of pathogenesis-related genes, RNA was extracted by the phenol-LiCl method according to Eggermont et al. [7]. RNA gel blot analysis was performed as described previously [16, 21]. All experiments are performed twice with similar results.

Acknowledgments. B.P.H.J.T. and I.A.M.A.P. are postdoctoral researchers of the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’, K.T.F.M. is the recipient of a predoctoral fellowship of the ‘Vlaams Instituut voor Bevordering van het WetenschappelijkTechnologisch Onderzoek in de Industrie’.

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